Dirofilaria immitis is the causative agent of heartworm disease in dogs, cats and wild canids, and is occasionally transmitted to humans resulting in pulmonary dirofilariasis. Prevalence values as high as 40% are found in some areas of the United States (Falls & Platt, American Journal of Veterinary Research, 43:738-739 (1982), and routine diagnostic screening has been recommended in an effort to control and manage the disease (American Heartworm Society, Proceedings of the Heartworm Symposium, 1992 (ed. Soll, M.D.), pages 289-294).
Traditionally, diagnosis relied exclusively on the detection of microfilariae in the blood. However, this method proved inadequate since a significant percentage of animals harbor occult infection, in which adult worms are present but there are no circulating microfilariae. More recently, commericial antigen test kits have become available which detect circulating female worm antigens. While these tests are highly specific, they have been shown to lack sensitivity for pre-patent or non-patent infections (Courtney, et al., Proceedings of the Heartworm Symposium, 1986 (ed. Otto, G.F.), pages 77-82; Dzimianski & McCall, Proceedings of the Heartworm Symposium, 1986 (ed. Otto, G.F.), pages 83-86; Wong & Fuller, Proceedings of the Heartworm Symposium, 1986 (ed. Otto, G.F.), pages 99-105; Courtney Journal of the American Animal Hospital Association, 24:27-32 (1988); Wong & Thomford, Journal of the American Animal Hospital Association, 27:33-38 (1991)).
Similar problems are encountered in diagnosis and management of the related filarial parasites of humans which are responsible for lymphatic filariasis (Brugia malayi, B. timori and Wuchereria bancrofti) and onchocerciasis (Onchocerca volvulus). However, a number of antigens from these parasites have now been identified which may be useful in diagnosis. Ov33-3 (Ov33) from O. volvulus, was one of the first filarial antigens reported to possess immunodiagnostic potential (Lucius, et al., Journal of Experimental Medicine, 167:1505-1510 (1988a)). The antigen was subsequently cloned by a number of investigators (Ov33.3 (Lucius, et al., Journal of Experimental Medicine, 168:1199-1204 (1988b)); Oc3.6 (Chandrashekar, et al., Journal of Clinical Investigation, 88:1460-1466 (1991)); OvD5B (Celine Nkenfou, Thesis, "Molecular Cloning of Genes Coding Antigens Specific For Onchocerca volvulus: Evlaution of Expressed Proteins For Use In The Diagnosis Of Onchocerciasis" University of Cameroon (1993)) and additional studies using recombinant antigen corroborated the earlier findings (Chandrashekar, et al., supra, Lucius, et al., Tropical Medicine and Parasitology, 43:139-145 (1992); Nkenfou, supra.) Perhaps surprisingly, homologs of this antigen have been found in other filarial parasites including B. malayi (Bm33) (Dissanayake, et al., Molecular and Biochemical Parasitology, 62:143-146 (1993)) and Acanthocheilonema viteae (Av33) (Willenbucher, et al., Molecular and Biochemical Parasitology, 57:349-351 (1993)). Interestingly, Bm33 was also shown to possess diagnostic potential for lymphatic filariasis (Dissanayake, et al., supra).
More recently, we described a 33 kDa antigen (DiT33) from D. immitis which reacts with sera generated against recombinant OvD5B, indicating the presence of a homolog in heartworm also (Mejia, et al., Parasite Immunology, 16:297-303 (1994)). A similar molecule exists in the intestinal nematode Ascaris suum since Ov33, Av33 (Willenbucher, et al., supra) and Bm33 (Dissanayake, et al., supra) share significant homology to an aspartyl protease inhibitor of A. suum Aspi3 (Martzen, et al., Biochemistry, 29:7366-7372 (1990)). However, the A. suum protein has not been assessed for diagnostic potential.
The inability to detect pre-patent infection with D. immitis limits surveillance and control activities, and results in delays in the evaluation of new therapies. Therefore a procedure for detecting early infection would be a useful tool in the management of heartworm infection.